Letters in Applied Microbiology 1997, 24, 269–275

Antimicrobial action of essential oils : the effect of dimethylsulphoxide on the activity of cinnamon oil P. Hili1, C.S. Evans and R.G. Veness School of Biological Sciences, University of Westminster, London, and 1Neal’s Yard (Natural Remedies) Ltd, London, UK 1233/96: received 17 July 1996 and accepted 28 August 1996 P . H IL I , C .S . EV AN S AN D R . G. VE N ES S. 1997. Fifty-one essential oils extracted from plants of known origin were tested for their antimicrobial activity against three bacteria, Pseudomonas aeruginosa, Staphylococcus aureus, Escherichia coli and four yeasts, Torulopsis utilis, Schizosaccharomyces pombe, Candida albicans and Saccharomyces cerevisiae using the drop diffusion method. All showed antimicrobial activity against at least one of the micro-organisms. Following this preliminary screening, 13 essential oils showing antimicrobial activity against at least five of the micro-organisms were tested in the range 50 mg ml−1 to 500 mg ml−1 using broth micro dilution techniques with dimethylsulphoxide (DMSO) as a dispersing solvent. The concentration of most of the oils required for total inhibition of growth was ×500 mg ml−1. Further studies on the antimicrobial action of cinnamon oil in the range 10–150 mg ml−1 showed that 50-fold higher activity was found when no dispersing solvent was used.

INTRODUCTION

Essential oils are the odorous, volatile products of plant secondary metabolism, normally formed in special cells or groups of cells or as glandular hairs, found on many leaves and stems. Oils occur as a globule or globules in the cell, and may also be excreted from cells lining the schizogenous ducts or canals. They may be present in such glandular cells or ducts in any or all organs of the plant including roots, stem, buds, leaves, flowers and fruits. However, oils are commonly concentrated in one particular region such as leaves, bark or fruit, and when occurring in various organs in one plant may possess different individual chemical components (Bonner 1991). The function of essential oils is believed to be largely communicative and a variety of complex interactions have evolved enabling plants to utilize essential oils to influence their environment. For example, the volatile monoterpenes that comprise about half of the oleoresin produced by certain species of conifer act as phytoprotective agents, defending the tree from herbivore and pathogen attack (Gijzen et al. 1991). Essential oils also act as antifeedants, with components of oils of shrubs and conifers inhibiting rumen micro-organCorrespondence to : Professor C. S. Evans, School of Biological Sciences, University of Westminster, 115 New Cavendish St, London W1M 8JS, UK. © 1997 The Society for Applied Bacteriology

isms upon which ruminants rely for digestion of plant material (van Beek 1986). The use of essential oils as antimicrobial agents has been described qualitatively for many years starting with the phenol coefficients of a number of essential oils published by Martindale (1910). Recently, it has been demonstrated that essential oils of female and hermaphrodite Thymus bacticus Boiss (thyme oil) showed marked activity against some Grampositive and Gram-negative bacteria and yeast. The activity was greater in the essential oils containing larger amounts of geraniol (Cruz et al. 1993). The antifungal activity of six essential oils was tested in vitro and 16 clinical isolates of the dermatophytes Epidermophyton floccosum, Microsporum canis, Trichophyton mentagrophytes and Trichophyton rubrum isolated from patients with dermatophytosis (Lima et al. 1992). The oils were obtained from the plants Annona classifora, Cinnamomum zeylanicum, Cymbopogon citratus, Ocimum gratissimum, Protium heptaphyllum and Xylopin frutesens. Oils (with the exception of that from P. heptaphyllum) caused inhibition of 81% of the studied strains of dermatophytes. Only two strains of T. rubrum and one of M. canis were resistant. The antimicrobial properties of oils from Melaleuca alternifolia (tea tree), Mentha piperita (peppermint) and Salvia officinalis (sage) were the most potent against facultatively anaerobic oral bacteria. Thymol and eugenol were the potent essential oil components (Shapiro et al. 1994). Within the

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literature there is some discordance between the level of antimicrobial activity reported for various essential oils. Data such as the geographical origin and the exact composition of the essential oils studied is rarely cited so that no consideration can be given to the basic question of what are the principles responsible for therapeutic action (Janssen et al. 1987). The importance of geographical conditions has been recently demonstrated in studies of two lemongrass species, Cymbopogon nardus (L.) and Cymbopogon pendulus grown under mild and moderate water stress for 45 and 90 d, that showed significant differences in the levels of the major oil constituents, geraniol and citral. The composition of the oils varied depending upon the level and duration of moisture stress (Singh-Sangwan et al. 1994). Comparison of methodologies has shown that agar diffusion and serial dilution methods to measure antimicrobial activity do not always give parallel results, confusing any assessment of antimicrobial activity (Youssef and Tawil 1990). Reproducibility is particularly difficult using the drop diffusion method (Carson and Riley 1995). Factors such as solubility and rate of vaporization of the oils are difficult to monitor and may lead to erroneous results. The aim of this study was to examine the antimicrobial activity of 51 essential oils of known geographical origin, against a range of Gram-positive and Gram-negative bacteria and yeasts. The effect of the addition of dimethylsulphoxide (DMSO) as an oil solubilizer on the antimicrobial activities was investigated. Furthermore, by determining the composition of each oil by gas chromatography the chemical components associated with the antimicrobial action could be identified.

Media

Oxoid nutrient broth (NB) and nutrient agar (NA) used for bacterial cultures were made up according to the manufacturer’s directions. Malt extract (Oxoid), 3 g, yeast extract (Oxoid), 3 g, glucose (BDH), 10 g, and mycologicol peptone (Oxoid), 5 g, were made up in 1 l of distilled water and autoclaved at 121 psi for 15 min (MYGP broth). MYGP agar was made up as the broth with the addition of 20 g l−1 of Oxoid Agar No. 3.

Micro-organisms

The micro-organisms used in this study were Staphylococcus aureus, NCTC 6571, Escherichia coli NCTC 10418, Pseudomonas aeruginosa NCTC 10662, Schizosaccharomyces pombe CMI 124, Saccharomyces cerevisiae CMI 61302, Candida albicans NCPF 113 and Torulopsis utilis CMI 23311. The bacteria were maintained at 4°C on slope cultures (NA) and the yeasts were maintained at 4°C on MYGP agar.

Antimicrobial activity study

To establish growth curves, organisms were transferred aseptically from stock slopes into 9 ml of broth and incubated at 30°C before dilution to 106 organisms ml−1. Spread plates were prepared using 200 ml of broth. A 5 ml drop of essential oil was placed in the centre of each plate and the plates incubated at 30°C for 24 h, after which the diameter of inhibition of growth in mm was measured. A minimum of three replicate plates was used for each oil.

MATERIALS AND METHODS Essential oils

Broth micro dilution assay

Fifty-one essential oils from a variety of commercial sources were tested. The species of plant, geographic origin and the part of the plant from which the oil was obtained are shown in Table 1.

The essential oils were prepared in DMSO. A stock solution of the oil to be tested was prepared by diluting 0·336 g in 20 ml of DMSO. Stock solution was then added to the culture broth to give final concentrations of 10–500 mg ml−1. To 2·0 ml of nutrient broth was added the required amount of oil in DMSO and 200 ml of the test bacteria diluted to 105 organisms ml−1. The samples were then incubated for 40 h at 37°C and the absorbances read at 700 nm. Control samples of broth and organism, and broth with DMSO plus organism (DMSO blanks) were incubated under the same conditions. To 2 ml of MYGP broth was added the required amount of oil in DMSO and 200 ml of the yeast in MYGP broth. The samples were incubated for 40 h at 30°C and the absorbances read at 700 nm. Control samples were incubated under the same conditions. The minimal inhibitory concentration (MIC) was defined as the lowest concentration which resulted in a reduction of ×90% in the observed absorbance.

Gas chromatography

To 400 ml of each essential oil was added 25 ml of isopentane and 25 ml of eicosane to act as low and high end markers respectively. The analyses were performed using a Perkin Elmer 8600 gas chromatograph fitted with flame ionization detector. A 50 m column 0·22 m i.d. with 1 mm of BP1 phase (SGE) was used ; oven temperature was 70° to 260°C at 2°C min−1 ; detector and injector temperatures were 300°C ; amount injected was 0·1 ml (split ratio 15 : 1) ; carrier gas was helium at a flow rate of 1·738 ml min−1 ; total run time was 95 min.

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Table 1 Botanical and geographical sources of essential oils

–—––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– Oils Species Part of plant Country of origin Extraction method –––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– — Aniseed Illicium verum Fruit China Steam distillation Basil Ocimum basilicum Herb India Steam distillation Bergamot Citrus bergamia Rind Ivory Coast Pressed Black pepper Piper nigrum Fruit India Steam distillation Cajuput Melaleuca cajiputi Twig/leaf Vietnam Steam distillation Cardamon Elettaria cardamomum Fruit Guatamala Steam distillation Cedarwood Cedrus atlantica Wood France Steam distillation Chamomile (blue) Matricaria chamomilla Flowers Egypt Steam distillation Chamomile roman Anthemis nobilis Flowers Hungary Steam distillation Cinnamon Cinnamomum zeylanicum Leaf Sri Lanka Steam distillation Clary sage Salvia sclarea Herb France Steam distillation Clove Eugenia caryophyllata Leaf Madagascar Steam distillation Coriander Couiandrum sativum Fruit Russia Steam distillation Cypress Cupressus sempervirens Needles France Steam distillation Eucalyptus Eucalyptus globulus Leaf France Steam distillation Eucalyptus citriadora Eucalyptus citriodora Leaf China Steam distillation Fennel Feniculum dulce Seed Spain Steam distillation Geranium Pelargonium graveolens Herb Egypt Steam distillation Ginger Zinger officinalis Root China Steam distillation Grapefruit Citrus paradisi Rind USA Pressed Ho leaf Cinnamonum comphora Leaf China Steam distillation Jasmine Jasminun officinalis Flowers Morocco Solvent extraction Juniper Juniperus communis Twigs/berries France Steam distillation Juniperberry Juniperus communis Berries France Steam distillation Lavender Lavendula hybrida Flowers France Steam distillation Lemon Citrus limonum Rind Italy Pressed Lemongrass Cymbopogon citratus Herb Nepal Steam distillation Lime Citrus aurantfolia Rind West Indies Pressed Mandarin Citrus reticulata Rind Brazil Pressed Marjoram Thymus masticina Herb Spain Steam distillation Melissa Melissa officinalis Herb France Steam distillation Myrhh Commiphora myrrha Resin Ethiopia Steam distillation Neroli Olibanum Orange Palmarosa Patchouli Peppermint Petigrain Pine Rose Rose absolute Rosemary Sage Sandalwood Tagettes Tea tree Thyme red

Citrus aurantium Boswellia thurifera Citrus sinensis Cymbopogon martini Pogostemon patchouli Mentha piperita Citrus aurantium Pinus sylvestris Rosa damascena Rosa damascena Rosmarinus officinalis Salvia officinalis Santalum album Tagettes glandulifera Melaleuca tenifolia Thymus vulgaris

Flowers Resin Rind Grass Leaves Herb Twigs/leaves Needles Petals Petals Herb Herb Wood Herb Leaves Herb

Morocco Somalia Corsica Indonesia USA India S. America France Morocco Morocco France Spain India Nepal Australia France

Steam distillation Steam distillation Steam distillation Steam distillation Steam distillation Steam distillation Steam distillation Steam distillation Steam distillation Steam distillation Steam distillation Steam distillation Steam distillation Steam distillation Steam distillation Steam distillation

Vetiver Vetuvera zizanoides Grass China Steam distillation Ylang ylang Canaga odorata Flowers Commores Steam distillation –––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– — © 1997 The Society for Applied Bacteriology, Letters in Applied Microbiology 24, 269–275

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Assay of cinnamon oil with and without DMSO as solubilizer against Saccharomyces cerevisiae

To 50 ml of MYGP broth in 250 ml conical flasks was added the required amount of cinnamon oil in DMSO (prepared as in the broth micro dilution assay). Parallel samples added the oil without DMSO to 50 ml MYGP broth. To each sample 5·0 ml of S. cerevisiae diluted to give 105 organisms ml−1 in the final solution were added. The samples were incubated upright for 48 h at 30°C on a rotating platform operating at 100 rev min−1. Following incubation, absorbance was measured at 700 nm and the mass of cells determined by centrifugation followed by freeze drying. Control samples of broth, broth¦organism, and broth¦oil were incubated under the same conditions. Analysis of data

For the drop diffusion method a minimum of three replicates was prepared. The mean and standard error were calculated. For the broth dilution method the mean absorbance of the triplicate samples was compared with the mean absorbance of the broth samples containing DMSO without oil to give a measure of the overall reduction in growth. For the comparative assay with and without DMSO, analysis of variation (ANOVA) was performed followed by Student t-tests. RESULTS AND D ISCUSSION

Of the 51 oils tested all showed activity in inhibiting the growth of at least one of the seven micro-organisms by the drop diffusion method. Thirteen oils demonstrating activity against at least five of the organisms are shown in Table 2.

The oils inhibited growth of the yeast species more than the bacterial species confirming previous work (Hammerschmidt et al. 1993). Pseudomonas aeruginosa proved to be the most resistant organism with only 13 of the 51 oils showing any inhibitory activity, which correlated with previous data (Panizzi 1993). Staphylococcus aureus proved to be the second most resistant organism, whereas 20 oils showed activity against E. coli. In drop diffusion experiments studying the inhibition of growth of the yeasts, 47 oils showed inhibitory activity against C. albicans, 49 oils showed activity against S. pombe and 50 showed activity against S. cerevisiae and T. utilis. The level of inhibitory activity of the oils varied from 0·3 to 90% of total growth. Analysis of each oil by gas chromatography showed that similar chemical constituents were found in some of these oils. A single chemical species, however, was not ubiquitously found suggesting that different constituents were contributing to the antimicrobial activity of each of the oils. The drop diffusion method proved useful as a preliminary screening test for such a large number of oils. However, because of its reliance on the solubility and the rate of diffusion of an oil in aqueous medium, low zones of inhibition could often falsely arise (Janssen et al. 1986). Further tests using broth dilution assays were made to ensure that the solubilizing agent itself did not have an inhibitory effect, or if so, that this effect was minimized. The concentration of DMSO used in the broth dilution assays was kept below 30 ml ml−1, selected from a preliminary screening of the level of DMSO to ensure the effects on bacterial and yeast growth were minimal. The results of the broth dilution assays are shown in Table 3. Above 90% reduction in growth was seen with clove oil

Table 2 Antimicrobial effect of essential oils using the drop diffusion method

–—––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– Oil E. coli Staph. aureus Ps. aeruginosa C. albicans S. cerevisiae T. utilis S. pombe –––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– — Clove 37·8 21·5 22·8 40·3 49·7 39·0 34·3 Coriander 13·2 11·7 6·0 28·7 32·0 37·0 33·0 Cinnamon 44·9 45·2 25·4 38·7 53·0 42·3 43·0 Cardamon 5·0 2·7 0 7·3 31·7 18·7 * Thyme 40·6 51·3 14·5 60·7 80·0 67·3 69·0 Tea tree 13·7 6·9 5·3 11·3 12·7 47·0 20·3 Marjoram 21·0 12·0 3·0 9·0 22·7 7·7 22·7 Ho leaf 16·0 15·5 4·7 37·3 40·0 34·0 47·7 Rosemary 1·3 0 0 6·7 12·3 9·7 16·3 Peppermint 16·1 16·0 4·8 16·0 13·0 13·3 17·0 Palmarosa 0 13·7 7·0 29·7 21·0 23·3 23·0 Lemongrass * * * * * 47·0 * Sage 15·0 5·3 0 11·0 9·0 12·7 15·7 –––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– — Mean diameter of inhibition of growth in mm, standard error within 10%. Maximum plate diameter was 80 mm. *Denotes patchy inhibition but no single clear zone. © 1997 The Society for Applied Bacteriology, Letters in Applied Microbiology 24, 269–275

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Table 3 % Reduction in growth caused by essential oils at 500 mg ml−1 in broth dilution tests for antimicrobial activity

–—––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– Oil E. coli Staph. aureus Ps. aeruginosa C. albicans S. cerevisiae T. utilis S. pombe –––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– — Clove 74·3 82·9 74·5 60·9 99·0 94·6 93·8 Coriander 67·3 44·2 74·3 74·7 67·8 86·6 17·9 Cinnamon 67·3 69·6 84·8 72·2 59·2 100 96·4 Cardamon 45·7 58·9 44·4 41·2 90·2 62·4 82·1 Thyme 75·2 94·5 77·2 100 100 100 100 Tea tree 27·8 43·3 75·4 37·1 68·9 32·7 74·2 Marjoram 0 29·1 14·3 19·8 93·7 12·2 71·1 Ho leaf 47·9 19·2 56·3 5·0 19·0 26·2 31·7 Rosemary 25·5 0·7 53·5 7·6 87·6 2·2 86·4 Peppermint 57·2 42·4 65·9 23·1 76·4 8·0 93·6 Palmarosa 42·2 46·6 63·2 70·9 100 100 100 Lemongrass 66·8 86·2 71·2 42·3 95·5 80·8 0 Sage 24·8 16·9 61·5 16·8 8·2 50·1 0 –––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– — Oils were solubilized in DMSO before addition to broth cultures.

against S. pombe, S. cerevisiae and T. utilis, cinnamon oil against S. pombe and T. utilis, cardamon against S. cerevisiae, thyme oil against Staph. aureus, S. pombe, S. cerevisiae, T. utilis and C. albicans, peppermint against S. pombe, palmarosa against S. pombe, S. cerevisiae and T. utilis, and lemongrass oil against S. cerevisiae. Clove, cinnamon and thyme oils caused ×50% reduction in growth with all organisms. No activity was observed with sage or lemongrass oils against S. pombe, and marjoram against E. coli. There was some variation in data from the drop diffusion assay compared with that from the broth dilution assay. For example, the diameter of inhibition in the drop diffusion test for cinnamon oil against S. cerevisiae and T. utilis was similar at 53 mm and 42·3 mm, whereas in the broth dilution assay, 59% and 100% reduction in cell growth was recorded respectively. Much higher concentrations of solubilized oils were required for inhibition than with the commercially available preservatives trichlorocarbanilide (TCC), trichlorohydroxydiphenyl ether (Irgasan DP 300) and hexachlorophene, all of which have values of MIC of ³0·1 ppm (Morris et al. 1979). Clove oil in DMSO had a MIC value of 300 ppm against S. cerevisiae and S. pombe and a MIC value of 400 ppm against T. utilis. Cinnamon oil showed a MIC value of 400 ppm against T. utilis and S. pombe. Thyme showed a MIC value of ³300 ppm for all the yeast species and palmarosa oil showed a MIC value of 400 ppm against S. pombe, S. cerevisiae and T. utilis. No MIC was demonstrated for any other oils at the concentrations tested. The activity of cinnamon oil against S. cerevisiae was markedly increased in the absence of DMSO (Table 4). Total inhibition of growth in broth dilution experiments occurred at a concentration of 10 mg ml−1 of cinnamon oil, compared with ×500 mg ml−1 when solubilized in DMSO. These

results showed an antagonistic effect of DMSO with cinnamon oil. Examination of the constituents of the 13 oils shown in Table 3 by reference to published data (Masada 1976), and separations achieved by gas chromatography showed common components which could contribute to the antimicrobial activity of these oils. Such components were identified as eugenol, linalool and p-cymene. Figure 1 shows a typical chromatographic separation of cinnamon oil. Eugenol was identified by gas chromatographic separations of oils from Thymus masticina (thyme), Cinnamomum zeylanicum (cinnamon) and Eugenia caraphyllata (clove), respectively. Morris et al. (1979) demonstrated MIC values of 500 ppm for pure eugenol against Staph. aureus, E. coli and C. albicans, which correlate closely with the MIC values obtained in this study with intact oils. Linalool, reported previously to have antiTable 4 Cell mass of Saccharomyces cerevisiae grown with

cinnamon oil, 48 h incubation — ––––––––––––––––––––––––––––––––––––––––––––––––––––– Cell mass (mg) –––––––––––––––––––––––––––––––––––– — −DMSO ¦DMSO Oil quantity — ––––––––––––––––– –––––––––––––––– — (mg ml−1) Mean S.D. Mean S.D. — ––––––––––––––––––––––––––––––––––––––––––––––––––––– 0 97·5 6·1 101·7 7·7 10 0 ND ND 50 0 64 8·5 150 0 26 7·0 500 0 0 — ––––––––––––––––––––––––––––––––––––––––––––––––––––– Means and standard deviations (S.D.) are for triplicate experiments. ND, Not determined.

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274 P . H IL I ET AL .

Eugenol

Eicosane

REFERENCES

75

50

25

0

Isopentane

of the oil, for good reproducibility of data. This study has shown much greater activity (50-fold more) can be attained with cinnamon oil against S. cerevisiae in the absence of solubilizer. This may be due to the partitioning of the oil between the aqueous phase and DMSO, distancing the oil from the cells. When no DMSO is used, the oil may be solubilized in the lipid membrane of the organism where it can have a greater effect on cell metabolism. The mechanism of action of essential oils or their components is unclear, as is the potential for resistance mechanisms arising to their antimicrobial activity. These aspects must be investigated if the use of essential oils as preservatives is to become economically viable.

Time (min)

Fig. 1 Separation of components of cinnamon oil by gas

chromatography on a BP1 column (50 m×0·22 mm), with flame ionization detection. Isopentane and eicosane were added to the oil as low and high end markers respectively

bacterial activity (Ross et al. 1980) was found in oils of Salvia officinalis (sage), Rosmarinus officinalis (rosemary), Thymus masticina (thyme), Cymbopogon citratus (lemongrass), Couiandrum sativum (coriander) and Cinnamomum comphora (ho leaf) in varying quantities. p-Cymene found in Melaleuca ternifolia (tea tree), Thymus vulgaris and Couiandrum sativum oils has antimicrobial activity (Kellner and Kober 1954). aPinene has been shown to have activity against Bacillus subtilis, E. coli, Staph. aureus, Proteus morganii, Ps. aeruginosa and is found in oils of sage, rosemary, tea tree, lemongrass, coriander, thyme, peppermint, and ho leaf (Katayama and Nagai 1960). Establishing the contribution of these components to the overall antimicrobial activity of any oil would require further investigation. The existence of a synergistic or antagonistic relationship between components may explain the differences in antimicrobial activity that arise for oils of the same species tested in different laboratories. In order to compare results of different studies the precise composition of oils must be known. The geographical origin of oils also needs to be considered in studies of antimicrobial activity. Kowal and Krupinska (1979) found large differences in the MIC values for essential oils of Thymus pulegioides from different regions in Poland, and the composition of essential oils from the same species grown in Southern Italy was influenced by the time of harvest (Senatore 1996). To date very few studies have comparatively examined the effects of oils with and without the use of solubilizer in liquid media. It is essential in such studies that the broth containing the oil is agitated thoroughly in order to achieve full dispersal

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Panizzi, L., Flamini, G., Cioni, P.L. and Morelli, I. (1993) Composition and antimicrobial properties of essential oils of 4 mediterranean Lamiaceae. Journal of Ethnopharmacology 39, 167–170. Ross, S., Elkeltawi, N.E. and Megalia, S.E. (1980) Antimicrobial activity of some Egyptian aromatic plants. Fitoterapia 51, 201– 206. Senatore, F. (1996) Influence of harvesting time on yield and composition of the essential oil of a thyme (Thymus pulegioides L.) growing wild in Campania (Southern Italy). Journal of Agriculture and Food Chemistry 44, 1327–1332. Shapiro, S., Meier, A. and Guggenheim, B. (1994) The anti-

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